See

discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/233929129

Isolation and Characterization of Proteins from

Chia Seeds (Salvia hispanica L.)
Article in Journal of Agricultural and Food Chemistry December 2012
DOI: 10.1021/jf3034978 Source: PubMed

CITATIONS

READS

27

912

2 authors, including:
Octavio Paredes-Lopez
Center for Research and Advanced Studies of t
268 PUBLICATIONS 4,959 CITATIONS
SEE PROFILE

Available from: Octavio Paredes-Lopez

Retrieved on: 20 September 2016

Article
pubs.acs.org/JAFC

Isolation and Characterization of Proteins from Chia Seeds (Salvia

hispanica L.)
Mara R. Sandoval-Oliveros and Octavio Paredes-Lopez*,

Programa de Posgrado en Alimentos del Centro de la Republica (PROPAC), Universidad Autonoma de Queretaro, Cerro de las
Campanas s/n, Santiago de Queretaro, Qro., Mexico CP 76010

Centro de Investigacion y de Estudios Avanzados del IPN, Unidad Irapuato, Km. 9.6 Libramiento Norte Carr, Irapuato-Leon,
Irapuato, Gto., Mexico CP 36821
ABSTRACT: Chia (Salvia hispanica L.) is a plant that produces seeds rich in some nutraceutical compounds with high protein
content, but little is known about them; for this reason the aim of this study was to characterize the seed storage proteins. Protein
fractions were extracted from chia seed our. The main protein fraction corresponded to globulins (52%). Sedimentation
coecient studies showed that the globulin fraction contains mostly 11S and 7S proteins. The molecular sizes of all the reduced
fractions were about 1550 kDa. Electrophoretic experiments under native conditions exhibited four bands of globulins in the
range of 104628 kDa. The denaturation temperatures of crude albumins, globulins, prolamins, and glutelins were 103, 105,
85.6, and 91 C, respectively; albumins and globulins had relatively good thermal stability. Selected globulin peptides by mass
spectrometry showed homology to sesame proteins. A good balance of essential amino acids was found in the seed our and
globulins, especially of methionine+cysteine.
KEYWORDS: Salvia hispanica, chia seed, proteins, globulins

utilized to elaborate coatings and edible lms.14 Although chia

is not a well-known food, its global production has increased in
recent years due to its healthy properties and new popularity.
Chia seeds are also used in the United States, Latin America,
and Australia as nutritional supplements, as well as in the
manufacture of bars, breakfast cereals, and cookies.8
On the other hand, protein isolates from vegetal sources are
of interest due to their increasing use as ingredients with
functional properties that can also improve the nutritive quality
of foods.15,16 The main proteins in seeds are storage proteins
accounting for about 6080% of the total proteins;17 their
analysis is greatly complicated by the polypeptides heterogeneity and the dierent solubility behaviors. Some studies have
reported that edible seeds generally contain two types of major
storage proteins that dier by size; the rst group includes
proteins with sedimentation coecients around 11S, which are
referred to as legumin-like or 11S globulins, and the second
group includes proteins with sedimentation coecients around
7S, which are classied as vicilin-like or 7S globulins. There is
also another type of proteins in a minor proportion, 2S-like
proteins.1820 Therefore, it is not surprising that the cereal seed
proteins have been a major topic of research for many years,
with the aim of understanding their structures, control of
synthesis, and role in the grain utilization as well as their
functional and nutraceutical properties.17 Up to now the
storage proteins of chia seed have not been fully characterized.
Thus, the aim of the present work was to fractionate and
characterize these proteins, as well as to identify, isolate, and

INTRODUCTION
Salvia hispanica L., with the popular name chia, is an annual
plant of the Lamiaceae family that grows in arid or semiarid
climates. Chia seed is considered a pseudocereal and, because
of its high oil content, is also an oilseed native of Mesoamerica,
exhibiting the greatest genetic diversity in the slope of the
Pacic Ocean from central Mexico to northern Guatemala.1
This oilseed and corn, beans, and amaranth were some of the
main crops for the pre-Columbian people.2 Mayans and Aztecs
used it as a medicine and food supplement for energy,
endurance, and strength needed under extreme conditions.1,3
Chia has been cultivated in Mexico for thousands of years, and
recent evaluations have shown that seeds have a large potential
to be exploited; their consumption may bring remarkable
benecial health eects.4 Seed composition appears very
attractive, being a good source of protein, with high amounts
of natural antioxidants such as phenolic compounds including
chlorogenic and caeic acids, quercetin, and kaempferol, as well
as high dietary ber content (>30% of the total weight).58 In
recent years these seeds have become increasingly important
for nutrition because of their high content of unsaturated fatty
acids; almost 60% is -linolenic acid (omega-3).9,10 All of these
mentioned features may provide health benets eective in
reducing cardiovascular diseases, obesity, regulation of intestinal
transit, and cholesterol and triglycerides levels, as well as
prevention of diseases such as type II diabetes and some types
of cancer.6,7,1012 On the other hand, protein content in chia
seeds is higher than most of the traditionally utilized grains;
they contain approximately 1923%, which is higher than
wheat (14%), corn (14%), rice (8.5%), oats (15.3%), and barley
(9.2%).2,13 Chia seed not only is a nutrient supplying food but
also has potential as a functional ingredient to be used as a
thickener in foods, and the mucilage from the seed has been
2012 American Chemical Society

Received:
Revised:
Accepted:
Published:
193

August 9, 2012
November 23, 2012
December 17, 2012
December 17, 2012
dx.doi.org/10.1021/jf3034978 | J. Agric. Food Chem. 2013, 61, 193201

Journal of Agricultural and Food Chemistry

Article

precipitate this fraction with 20% of trichloroacetic acid (TCA). A total

of 20 g of each protein sample was loaded per lane, and approximate
molecular sizes of the proteins were determined by Invitrogen
molecular size standards. Electrophoresis was performed at a constant
current of 60 V per gel for approximately 2 h. The gels were stained
with Coomassie brilliant blue R.
Thermal Characterization of Protein Fractions. Triplicate
samples (5 mg of each protein isolate) were suspended in 15 L of
water and hydrated for 24 h prior to the test. A hermetic DSC pan was
used to encapsulate the samples of freeze-dried protein dispersed in
deionized water. The denaturation temperature of protein fractions
was measured using a Q1000 dierential scanning calorimeter (DSC)
(TA Instruments, New Castle, DE, USA). The sealed pan was placed
in a calorimeter previously calibrated with indium. The temperature
scan was carried out from 20 to 180 C at 10 C/min.15,27 Universal
Analysis 2010 Software (TA Instruments) was used to analyze the
thermograms to determine the denaturation peak temperature (Td),
denaturation temperature range (Td), and denaturation enthalpy
(Hd).
Mass Spectrometric Analysis. Tandem Mass Spectrometry (LC/
ESI-MS/MS). To further characterize the isolated globulin proteins,
peptide mass ngerprinting was performed for some bands with more
intensity and clarity, obtained in the SDS-PAGE. Bands from the
electrophoresis were carefully excised from the gel and washed
successively with ultrapure water and 25 mM ammonium bicarbonate
(NH4HCO3). Gel pieces were dehydrated with acetonitrile (ACN) to
remove contaminants and stain. Samples were reduced with 10 mM
dithiothreitol (DTT) in 25 mM NH4HCO3 followed by protein
alkylation with 55 mM iodoacetamide. The isolated proteins were
digested with modied porcine trypsin (Promega, Madison, WI, USA)
and extracted from the polyacrylamide gel. The pooled supernatants
were concentrated, and the peptides were desalted and concentrated
to a nal volume of 5 L with Zip-Tip C18 (Millipore, Billerica, MA,
USA), according to the manufacturers protocol.28
MS analysis was carried out on a 3200 Q TRAP hybrid tandem
mass spectrometer (Applied Biosystems/MDS Sciex, Concord, ON,
Canada), equipped with a nanoelectrospray ion source (NanoSpray II)
and a MicroIonSpray II head. The instrument was coupled online to a
nano Acquity Ultra Performance LC system (Waters Corp., Milford,
MA, USA). Mass calibration of the hybrid triple-quadrupole linear ion
trap spectrometer was done with polypropylene glycol standard
solutions. The instrument was then tuned and tested using [Glu1]brinopeptide B (Sigma-Aldrich). Peptides were separated on a BEH,
C18 UPLC column (1.7 m, 75 m 100 mm, Waters Corp.)
equilibrated with 2% acetonitrile and 0.1% formic acid, using a linear
gradient of 270% acetonitrile, and 0.1% formic acid over a 60 min
period, at a ow rate of 0.25 L/min. Spectra were acquired in
automated mode using information-dependent acquisition (IDA).
Precursor ions were selected in Q1 using the enhanced MS mode
(EMS) as a survey scan. The scan range for EMS was set at m/z 400
1500 and 4000 amu/s, with an ion spray voltage of +2.2 kV applied to
a Picotip emitter FS150-20-10-N (New Objective, Woburn, MA,
USA). The interface heater for desolvation was held at 150 C. The
precursor ions were fragmented by collisionally activated dissociation
(CAD) in the Q2 collision cell. Collision voltages were automatically
adjusted on the basis of the ion charge state and mass using rolling
collision energy. Generated fragments ions were captured and their
masses analyzed in the Q3 linear ion trap.
Database Search and Protein Identication. Data interpretation
and protein identication were performed with the MS/MS spectral
data sets using the MASCOT search algorithm (version 1.6b9, Matrix
Science, London, UK; available at http://www.matrixscience.com).
Searches were conducted using the SwissProt database of the National
Center for Biotechnology Information nonredundant database
(NCBInr, http://www.ncbi.nih.gov, and http://genetics.bwh.harvard.
edu/msblast).
Amino Acid Analysis. Amino acid content was determined in
triplicate using an RP-HPLC with precolumn derivatized phenylisothiocyanate, according to previously published procedures.29,30 In
brief, the dried protein samples were hydrolyzed, in triplicate (1 mg

characterize the main protein fraction from molecular, thermal,

and nutritional viewpoints with the aim of generating
information leading to a wider use of these macromolecules.

MATERIALS AND METHODS

A single lot of chia seeds (S. hispanica L. var. Chionacalyx) harvested in

November 2010 was provided by Ing. Roberto Nahum Amaya
Zamora, from Colima (Mexico). The chemicals were purchased from
Sigma-Aldrich (St. Louis, MO, USA) and the reagents used for
electrophoresis and staining solutions from Bio-Rad (Hercules, CA,
USA). Molecular weight standards were purchased from Invitrogen
(Mexico, DF, Mexico) and amino acid standards from Pierce
(Rockford, IL, USA). All chemicals used were of analytical grade,
and deionized water was used.
Chemical Analyses. Moisture, fat, protein (N 6.25), ash, and
total dietary ber contents were determined using standard methods
925.10, 920.85, 981.10, 923.03, and 985.29, respectively, reported by
AOAC;21 the total nitrogen content of the our was analyzed using the
micro-Kjeldahl procedure to determine the crude protein content.
Sample Preparation. The seeds were soaked in water (ratio 1:10,
w/v) during 2 h. The seeds, which are coated with swollen mucilage,
were frozen (80 C) overnight and freeze-dried, and the dry
mucilage was removed mechanically.5 Mucilage-free seeds were milled
into a our and passed through a 0.5 mm mesh to obtain a uniform
particle size. The our was defatted with hexane (ratio 1:10, w/v) in a
Soxhlet unit at 6570 C and dried overnight under a hood at room
temperature to remove the trace of remaining hexane; then a second
grinding was performed to obtain a smaller particle size (0.18 mm),
and afterward it was stored at 4 C until use.6
Protein Extraction and Fractionation Procedure. Fractionation of proteins was carried out according to the Osborne22
classication using a modication of the method reported by Barba
de la Rosa et al.23 All of the suspensions were stirred for 4 h at 4 C
and centrifuged at 14000g during 1 h at 4 C; the rst suspension was
our/water (1:10, w/v), and the resulting supernatant was designated
as crude albumin fraction. The pellet was resuspended in 10 mL of a
50 mM Tris-HCl, pH 8, buer solution containing 0.5 M NaCl. After
centrifugation, the supernatant was separated, and it was referred to as
globulin fraction; the pellet was resuspended in 10 mL of a 70%
aqueous isopropanol solution and extracted under constant stirring.
The resulting supernatant was now the prolamin fraction, and the
pellet was resuspended in 10 mL of a 0.1 M Na2B4O710H2O, pH 10,
solution. After centrifugation, the supernatant was separated, the
glutelin fraction was obtained, and the pellet was the residue. The
residue after extraction from each solvent was washed twice using a
small portion of water. The washings and the rst extract were
combined for each fraction. Fractions were dialyzed, freeze-dried, and
stored at 4 C for further analysis.
Protein Quantication. The protein content of the isolated
fractions was assessed by micro-Kjeldahl, and the soluble protein
content in each fraction was determined by the BCA (Pierce)
method.21
Sedimentation Coecient Determination of Globulin
Fraction. The globulin isolate fraction was layered onto a linear
sucrose density gradient (520% in a pH 8 buer of 50 mM Tris-HCl
+ 0.3 M NaCl) and centrifuged at 218000g during 24 h at 4 C
(Beckman, 15-65, SW 40 ti rotor). Fractions of 1 mL were collected,
and protein concentration was determined according to the BCA
method.24
Molecular Size Determination. Molecular size was determined
by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDSPAGE) according to the Laemmli method.25 SDS-PAGE was carried
out on a slab gel (5% stacking gel, 12% separating gel), in an SDS
Trisglycine discontinuous buer system. Also, for a better resolution
of proteins with low molecular weight, the method of Schagger and
Von Jagow26 was used with polyacrylamide gradient gels of 513%.
Proteins were prepared in native, reducing, and nonreducing
conditions, in buer solutions with or without 2-mercaptoethanol.
To improve the electrophoretic pattern of prolamins, we have to
194

dx.doi.org/10.1021/jf3034978 | J. Agric. Food Chem. 2013, 61, 193201

Journal of Agricultural and Food Chemistry

Article

each), in constant-boiling 6 N HCl, and melted crystalline phenol was

added for aromatic amino acid protection. Hydrolysis was performed
under vacuum in a heating block for 24 h at 110 C. After cooling at
room temperature, the samples and mixture of amino acid standards
were derivatized by adding 20 L of solution containing ethanol/
water/triethylamine/phenylisothiocyanate (7:1:1:1, v/v) and incubated at room temperature for 20 min. The samples were dried in a
vacuum centrifuge, dissolved in 0.2 mL of 50 mM sodium phosphate
buer, pH 7.4, and ltered through a 0.22 m lter, and then the
sample was subjected to reverse-phase chromatography. The phenylthiocarbamyl derivatives were detected by their absorbance at 254 nm.
After separation, the peaks were integrated and quantied using a
standard curve of peak areas previously obtained from known
concentrations of the amino acid standard mixtures.
Chemical Score. The chemical score (CS) was calculated as
m
CS = EAA 100
mR

Table 2. Proportion of the Protein Fractions of Chia Seed

sample
albumins
globulins
prolamins
glutelins
insoluble proteins
a

where mEAA is the mass (g) of the essential amino acid in the examined
protein and mR is the corresponding reference amino acid requirements. The FAO/WHO/UNU31 pattern of amino acid requirements
for the two extreme age groups (0.51 and >18 years) was used as a
reference to calculate amino acid scores and assess the quality of
dietary protein.
In Vitro Digestibility. The in vitro digestibility method used was a
modication of that of Hsu et al.32 Fifty milliliters of an aqueous
protein suspension (6.25 mg protein/mL) was prepared; these
solutions were adjusted to pH 8.0 with 0.1 N HCl or NaOH. On
the other hand, a multienzyme solution was also prepared (1.6 mg
trypsin with 15 units per mg powder; 3.1 mg chymotrypsin with 60
units per mg powder; and 1.3 mg peptidase with 40 units per g
powder/mL) which was adjusted to pH 8.0 and maintained in an ice
bath until use. Five milliliters of the multienzyme solution was added
to the protein suspension with agitation, which was then incubated at
37 C in a water bath with stirring during 10 min. A rapid decline in
pH was produced at 10 min; for this reason the pH drop was recorded
in this period to estimate the in vitro digestibility.
Statistics. All statistical analyses were performed using SigmaPlot
statistical software (version 11.0). All experiments were conducted at
least in triplicate, and data are expressed as the mean standard
deviation (SD).

g/100 g proteina
17.3
52.0
12.7
14.5
3.4

0.8
1.0
0.2
0.2
0.6

Values are the mean SD of three determinations.

Figure 1. Electrophoretic patterns of protein fractions from defatted

chia our, in reduced conditions with the presence of mercaptoethanol. Lanes: 1, molecular weight marker; 2, albumins; 3, globulins; 4,
prolamins; 5, glutelins.

RESULTS AND DISCUSSION

Proximate Analysis. The proximate composition and
dietary ber of chia seed are summarized in Table 1. The

Table 1. Proximate Composition and Dietary Fiber of Chia

Seed
component
moisture
lipids
protein
ash
dietary ber
soluble
insoluble
total
carbohydrates (by dierence)
a

amounta (g/100 g dry solids)

4.5
32.5
22.7
3.7

0.0
2.7
0.7
0.3

Figure 2. Sedimentation coecient prole of globulins from chia

seeds: graph of protein concentration versus sedimentation coecient.

8.2 0.8
25.4 2.2
33.5 2.7
3.1

(13.4%), wheat (12.6%), and soybean (15%). This conrms

that chia is an outstanding source of dietary ber as compared
to most known sources.34 On the other hand, the oil content
(32.5%) was higher than that of other oilseeds of commercial
importance, such as soybean (24%) and cotton seed (22
24%).9 The protein content was similar to that of lentil (23%)
or chickpea (21%) and higher than that of chan (14%), of the
same family, and other oilseeds.35,36 Thus, chia is an important
source of protein; this, together with the high content of oil rich
in omega-3, makes the potential of this seed, for health and
nutrition, of a very remarkable level.

Values are the mean SD of three determinations.

values of all measured parameters are consistent with those

reported by Ayerza and Coates2 and Reyes-Caudillo et al.33 The
seeds contain low amounts of moisture (4.5%), minerals
(3.7%), and carbohydrates (3.1%), as well as a large amount of
total dietary ber (33.5%), which is superior to traditional
sources of ber such as axseeds (22.3%), barley (17.3%), corn
195

dx.doi.org/10.1021/jf3034978 | J. Agric. Food Chem. 2013, 61, 193201

Journal of Agricultural and Food Chemistry

Article

Figure 3. Electrophoretic patterns of the globulin fraction from chia seeds: (A) native conditions (lane 1, molecular weight marker; lane 2, native
globulins); (B) SDS-PAGE of globulins in the presence and absence of mercaptoethanol (lane 1, molecular weight marker; lane 2, presence of
mercaptoethanol; lane 3, absence of mercaptoethanol).

Fractionation and Quantication of Chia Seed

Proteins. The total protein content of defatted our of
mucilage-free chia seeds increased to 35.5% as determined by
Kjeldahl analysis (data not shown). Hereinafter we continued
working with defatted our of mucilage-free seeds. After
protein extraction and fractionation by solubility, each fraction
was quantied by micro-Kjeldahl and BCA method, and the
proportion obtained was 17.3% of crude albumins, 52% of
globulins, 12.7% of prolamins, and 14.5% of glutelins, whereas
3.4% of the protein was not recovered (Table 2). This pattern
of protein composition shows some similarity with other
important seeds such as peas, lupins, and cotton.37 It is clear
that these seed proteins may vary according to the botanical
source, type of variety, preparation of the meal, extraction
method, and other factors. However, the higher proportion
found here for the chia globulin fraction is consistent with
previous studies,6 which have reported levels of 64.8%.
Electrophoretic Pattern of Osborne Fractions. In the
electrophoretic analysis by SDS-PAGE (Figure 1) we found
that the fractions of albumins and globulins showed a large
number of bands with a wide range of molecular sizes; however,
in the albumin fraction we did not observe bands with high
intensity, unlike the globulin fraction, which showed seven
concentrated bands with increased presence having molecular
sizes between 18 and 35 kDa. As indicated before, to slightly
improve the electrophoretic pattern of prolamins, it was
necessary to precipitate the protein with TCA due to the low
resolution of this fraction; at the end, only three bands between
25 and 33 kDa were visible. On the other hand, the glutelin
fraction showed four bands with molecular sizes around 2030
kDa with a certain similarity to globulins; this is consistent with
the classication of Fukushima,38 who included these two
fractions in a single one, based on primary structur homology
criteria.
Determination of Sedimentation Coecient of Globulins. The sedimentation prole of the globulin fraction from

chia seed on sucrose density gradient revealed the presence of

four protein fractions as shown in Figure 2, conrming that 11S
globulin was a major component of this fraction. It also showed
the presence of 7S-like proteins in a much lesser proportion,
which is common in dicotyledon seeds. To our knowledge, this
is the rst time that chia seed globulins have been characterized
by the sedimentation coecient; thus, it is not possible to make
comparisons with other studies on chia globulins. However, the
sedimentation pattern of chia seeds is somewhat similar to that
observed in globulins of amaranth, sesame, barley, and some
other seeds.3941 The presence of the globulins 7S and 11S in
food ingredients may confer nutritional, physiological, and
functional characteristics to the foods that are dependent on
their structural sequence and conformation as well as on their
physicochemical properties; for example, the 7S-like proteins in
general exhibit emulsifying properties, the 11S globulins of
amaranth have peptides with antihypertensive activity, and the
11S-like proteins from various sources possess good gelling
capacity; in other words, these proteins may act as ingredients
providing favorable characteristics to food products.23,39
Figure 2 also reveals the presence in a low proportion of
proteins with unusual sedimentation coecients such as 6S and
19S; this may be because the subunits of 11S globulins
sometimes form intermediate structures of high molecular
weight.42 These proteins were also seen in the electrophoretic
pattern under native conditions in Figure 3A represented by the
bands of 104 kDa (6S) and 628 kDa (19S); these last values of
the sediment constants may be due to dierent aggregation
disaggregation phenomena during the preparation procedure of
the globulins (i.e., temperature, pH, dialysis, lyophilization),
and especially the pH is involved in the structural changes of
the globulins, producing association and dissociation of the
hexamer subunits of this protein fraction.18,43
Molecular Size Determination of Globulin Fraction.
The electrophoretic pattern of the globulin fraction in native
conditions (Figure 3A) showed four bands with molecular sizes
196

dx.doi.org/10.1021/jf3034978 | J. Agric. Food Chem. 2013, 61, 193201

Journal of Agricultural and Food Chemistry

Article

Figure 4. DSC thermographs of the four protein fractions of chia seed our: (A) albumins; (B) globulins; (C) prolamins; (D) glutelins.

between 50 and 60 kDa; these monomers under reducing

conditions with -mercaptoethanol are resolved into acidic (30
kDa) and basic (20 kDa) subunits.23,39 These data t with the
results observed in the electrophoretic pattern of globulins in
Figure 3B, results that under reducing and nonreducing
conditions indicate that the globulin fraction contains disulde
bonds in their structure, ensuring the abundant presence of 11S
protein.
Thermal Characterization of Protein Fractions. The
thermal properties of the protein fractions from chia seed our
were analyzed by DSC; this is a valuable tool for assessing the
potential of protein isolates as functional ingredients in
dierent food systems requiring heat processing. Because the
functional properties of proteins are greatly inuenced by their
conformation and DSC is a technique highly sensitive to
conformational changes,44 we applied it to our protein
fractions, and the thermograms are shown in Figure 4. There
was only a single peak for each of the fractions; peaks for
albumins and prolamins appeared to have a better denition
than those for the other fractions. Table 3 shows thermal

Table 3. Denaturation Temperature Range (Td),

Denaturation Peak Temperature (Td), and Denaturation
Enthalpy (Hd) of Lyophilized Extract of the Protein
Fractions of Chia Seed

fraction

Td (C)

albumins
globulins
prolamins
glutelins

96.0118.8
94.3116.6
72.193.2
76.0104.9

Tda (C)
103.6
104.7
85.6
91.3

0.7
0.2
0.6
0.8

Hda (J/g)
12.6
4.7
2.3
6.2

0.8
0.9
0.2
0.1

Values are the mean SD of three determinations.

between 104 and 628 kDa, of which the major band was that of
383 kDa and represents the 11S protein, thereby conrming
that the results obtained in the determination of sedimentation
coecient and the molecular size are consistent with those
reported in the literature of approximately 300400 kDa. This
corresponds to the hexameric conformation typical of the 11S
proteins, which are resolved in denaturing conditions without
-mercaptoethanol into monomers with molecular sizes
197

dx.doi.org/10.1021/jf3034978 | J. Agric. Food Chem. 2013, 61, 193201

Journal of Agricultural and Food Chemistry

Article

the protein structure; the values for albumin, globulin,

prolamin, and glutelin were 12.6, 4.7, 2.3, and 6.2 J/g,
respectively. These results demonstrate how dierent ionic
strengths may be aecting the stability of chia proteins because
the thermal stability is mainly controlled by the balance of polar
and nonpolar residues in a protein structure, and a higher
content of nonpolar residues means greater thermal stability. 24,27 The relatively low enthalpy values and high
denaturation temperatures found for chia proteins deserve
further studies. Due to the thermostability of proteins found in
chia seeds, they may be used in food systems undergoing high
heat treatments.
Mass Spectrometric Analysis. To identify peptides in
globulins of chia seed and to conrm the results obtained by
the analysis of sedimentation coecient, we used liquid
chromatography electrospray ionization quadrupole time ofight tandem mass spectrometry analysis (LC-ESI-Q/TOF
MS/MS). Nine major protein bands from one-dimension
electrophoretic gel (Figure 5) were selected for further
identication by MS/MS. The results of this analysis are
shown in Table 4; this conrmed the presence of peptides
belonging to the 11S protein (G3, G4, G5, G6) and, in less
proportion, peptides of the 7S protein (G1, G2). Only three of
nine analyzed peptides could not be identied by this method
(G7, G8, G9). Protein identication by studying homologous
sequences or comparison of their mass can be carried out on
the basis of the fact that many proteins from plants are highly
conserved. Thus, proteins that share sequence similarity are
likely to play the same function; for this reason, the databases
tend to identify homologous sequences in dierent species that
may facilitate the identication and allocation of function. In
this study, all of the proteins that were identied showed
homology to proteins of sesame (Sesamum indicum) with a low
rate of coverage; however, this is common in the protein
identication of species for which the genome has not been
sequenced, and even in these cases is dicult to obtain
homologies.20 The fact that all of our identied proteins have
homology with sesame proteins leads us to the general belief
that the synthesis and behavior of the chia seed proteins may
have, like the sesame seeds proteins, benecial eects such as
lowering blood pressure and improvement of cholesterol
proles.46
Amino Acid Analysis. The amino acid composition of
defatted our showed that chia seeds are a good source of
sulfur, aspartic, and glutamic amino acids (Table 5). The prole
of amino acids in chia seeds has been reported previously by
Ayerza and Coates,13 which is in general agreement with that of
this study. On the other hand, the composition of amino acids

Figure 5. Electrophoretic pattern of globulin fraction from chia seeds

by the Shagger and von Jagow26 method in polyacrylamide gradient
(513%). Lanes: 1, molecular weight marker; 2, globulins in the
presence of mercaptoethanol.

properties of the fractions. The onset and end temperatures

indicate that the protein starts to denature or unfold and
completely denatures, respectively, whereas the midpoint of the
peak is considered as the Td, the denaturation temperature of
the protein; as mentioned before, this is clearer in the
thermograms of albumins and prolamins (Figure 4). The
denaturation temperatures of albumins and globulins were
similar, 103.6 and 104.7 C, respectively. These denaturation
temperatures were relatively higher than those for several plant
proteins, such as legumes and cereals, which are mostly lower
than 100 C;45 additionally, the Td of the four fractions from
amaranth seeds are between 70 and 96 C.24 The fact that in
the thermograms is observed only a single peak of denaturation
suggests the presence of a single proteinic species. Moreover,
due to the high denaturation temperatures, especially of the
albumins and globulins, it is most likely that the conformation
of the components of these fractions are stabilized by a number
of hydrophobic interactions, which are of endothermic nature
and therefore require a high amount of energy for
denaturation.24 On the other hand, the H values give
information about the amount of energy required to denature
Table 4. Globulin Proteins Identied by LC-MS/MS

band

theor MWa/pIb

protein identity

G1
G2
G3
G4
G5
G6
G7
G8
G9

67027/7.55
67027/7.55
56553/8.57
56553/8.57
56553/8.57
56553/8.57

7S globulin
7S globulin
11S globulin
11S globulin
11S globulin
11S globulin

organism
Sesamum
Sesamum
Sesamum
Sesamum
Sesamum
Sesamum

indicum
indicum
indicum
indicum
indicum
indicum

peptides matched
5
6
4
4
6
5
unidentied
unidentied
unidentied

sequence coverage (%)

score

NCBI accession no.

4
8
5
8
10
10

71
66
110
112
139
134

gi|13183177
gi|13183177
gi|13183173
gi|13183173
gi|13183173
gi|13183173

MW, theoretical molecular weight. bpI, theoretical isoelectric point.

198

dx.doi.org/10.1021/jf3034978 | J. Agric. Food Chem. 2013, 61, 193201

Journal of Agricultural and Food Chemistry

Article

Table 5. Amino Acid Composition of Chia Defatted Flour and of the Globulin (glob) Fraction and Contribution of Essential
Amino Acids with Respect to the Requirement Patterns for Two Age Groups
contribution (%) of essential amino acids
infants 0.51 year

amino acid

seed our

Asp
Glu
Ser
Gly
Arg
Ala
Pro
Hisa
Thra
Vala
Met+Cysa
Ilea
Leua
Phe+Tyra
Lysa

47.3
70.8
26.2
22.8
42.3
26.8
19.9
13.7
18.0
28.5
27.8
24.2
41.5
38.8
29.9

0.9
1.1
0.3
0.7
0.4
0.3
0.7
0.1
0.2
0.4
0.5
0.4
0.6
0.5
0.5

globulins
72.9
243.0
69.3
73.6
94.2
39.4
106.4
40.0
62.3
35.9
57.5
30.1
44.4
109.3
15.4

adults >18 years

%CRc

amino acid content (mg/g raw protein)

0.4
1.3
0.7
0.6
1.6
0.5
1.0
0.6
0.7
0.6
0.4
1.2
1.7
0.8
0.6

%CR

RP

seed our

glob

RP

seed our

glob

20
31
43
28
32
66
52
57

69
58
66
99
76
63
75
52

200
201
83
205
94
67
210
27

15
23
39
22
30
59
38
45

91
78
73
126
81
70
102
66

267
271
92
261
100
75
288
34

a
Essential amino acids. bRP, requirement patterns for the dierent age groups (mg/g raw protein). c%CR, coverage of requirement for that specic
essential amino acid in percentage.31

fraction isolation (Table 5). It is interesting to point out that

the percentage of essential amino acids quantitated in Table 5 is
about 50% (46.5%), which is much higher than the
corresponding values reported for soybean (41.0%) and
saower (38.1%); this is an important aspect in favor of the
quality of the chia seed proteins.47
In Vitro Digestibility. The results of in vitro digestibility
analysis are shown in Table 6. The in vitro digestibility of the
globulin fraction (82.5%) was slightly higher than that of the
defatted chia our (78.9%), but slightly lower than that for
casein (88.6%) used as control. The in vitro digestibility of
defatted chia our showed a value similar to that previously
reported for chia; these values are around 77.5%,6 and they are
also similar to those reported for Phaseolus vulgaris (77.5%) and
higher than those for some cereals such as maize (66.6%), rice
(59.4%), sorghum (59.1%), and wheat (52.7%).48,49 There are
no reports of antinutritive factors in chia, ruling out the
presence of protease inhibitors that could retard the in vitro
digestibility.49 This digestibility value is a general indicator of
the nutritional quality of proteins, and it may be associated with
their special arrangement, because the tertiary and quaternary
structures have dierent susceptibilities to proteolytic
enzymes.50
In conclusion, chia seeds show high contents of proteins and
ber, particularly insoluble ber and lipids. Globulins were by
far the major fraction with seven intense bands between 18 and
35 kDa, four of them with some similarity to those of glutelins.
Ultracentrifugation experiments showed that globulins contain
11S and 7S proteins as major and minor components.
Electrophoretic studies under reducing and nonreducing
conditions conrmed the presence of 11S type of proteins.
Thermal stability using DSC showed that albumins and
globulins have denaturation temperatures above 100 C, higher
than those from other plant proteins; these protein fractions
may be suitable for certain food products undergoing high heat
treatment. Mass spectrometry analysis identied four major
globulin peptides as belonging to 11S type of proteins and two

Table 6. In Vitro Digestibility of Flour and Globulin

Fraction of Chia Seed

sample

digestibilitya (%)

globulins
defatted our
casein

82.5 1.1
78.9 0.7
88.6 1.1

Values are the mean SD of three determinations.

of the isolated globulin fraction has a high content of aromatic

and sulfur amino acids as well as threonine and histidine; this
sample also exhibited a high percentage of glutamic and aspartic
acids, which is typical in seeds with an abundance of globulins.
Low levels of lysine were observed in both samples. The
abundance of sulfur amino acids suggests that they may be
intimately involved in maintaining the tertiary and quaternary
structure of the proteins, and the presence of high levels of
glutamic acid has been of interest in the food industry due to
the potential of this amino acid to stimulate the central nervous
and immunologic systems in humans.47 The potential of
aspartic acid rich foods in the hormonal regulation for the
proper functioning of the nervous system has been reported. In
general, the protein quality of chia has been demonstrated to be
higher than that of some cereals and oilseeds; this may
represent an important nutraceutical contribution to foods that
contain chia seeds and isolated globulins as ingredients.18
Chemical Score. In the case of the seed our, the coverage
of the amino acid requirement for infants was about 100%
satisfactory for the sulfur amino acids; the coverage for the
remaining essential amino acids ranged from 52 to 76%. On the
other hand, the required coverage was much better for adults;
the essential amino acids in seed our varied from 66 to 126%.
The globulin fraction exhibited ranges of coverage of
requirements wider than those of seed our, 27210% for
infants and 34288% for adults; the lowest values corresponded to lysine in view of the known limitations of cereals in
this amino acid and its partial destruction during the protein
199

dx.doi.org/10.1021/jf3034978 | J. Agric. Food Chem. 2013, 61, 193201

Journal of Agricultural and Food Chemistry

Article

of them as 7S. The essential amino acids of both seed our and
globulin exhibited in general a relatively good balance of them,
especially Met+Cys; globulins are a good source of aromatic
amino acids. The in vitro digestibility of seed our and
globulins reached better values than those reported for most
cereals. In brief, our results support the potential use of chia
seeds as a good source of proteins, including fractions with
remarkable thermal stability, and other important nutritious
and nutraceutical components.

(8) Martnez, L.; Marn, M.; Salgado, C.; Revol, J.; Penci, M.; Ribotta,
P. Chia (Salvia hispanica L.) oil extraction: study of processing
parameters. Food Sci. Technol. 2012, 47, 7882.
(9) Ayerza, R. Oil content and fatty acid composition of oil of chia
(Salvia hispanica L.) from five locations in northwestern Argentina. J.
Am. Oil Chem. Soc. 1995, 72, 10791081.
(10) Jin, F.; Nieman, D. C.; Sha, W.; Guoxiang Xie, G.; Qiu, Y.; Jia,
W. Supplementation of milled chia seeds increases plasma ALA and
EPA in postmenopausal women. Plant Foods Hum. Nutr. 2012, 67,
105110.
(11) Bemelmans, W.; Broer, J.; Fesfens, E.; Smit, A.; Muskiet, F.;
Lefrandt, J.; Bom, V.; May, V.; Meyboom-de Jong, B. Effect of an
increased intake of -linolenic acid and group nutritional education on
cardiovascular risk factor: the Mediterranean -linolenic enriched
Groningen dietary intervention study. Am. J. Clin. Nutr. 2002, 75,
221227.
(12) Poudyal, H.; Panchal, S.; Waanders, J.; Ward, L.; Brown, L.
Lipid redistribution by -linolenic acid-rich chia seed inhibits stearoylCoA desaturase-1 and induces cardiac and hepatic protection in diet
induced obese rats. J. Nutr. Biochem. 2012, 23, 153162.
(13) Ayerza, R.; Coates, W. Protein content, oil content and fatty
acid profiles as potential criteria to determine the origin of
commercially grown chia (Salvia hispanica L.). Ind. Crops Prod.
2011, 34, 13661371.
(14) Munoz, L. A.; Cobos, A.; Diaz, O.; Aguilera, J. M. Chia seeds:
microstructure, mucilage extraction and hydration. Int. J. Food Eng.
2012, 108, 216224.
(15) Paredes-Lopez, O.; Ordorica-Falomir, C.; Guevara-Lara, F.;
vegetales: presente y futuro de la
Covarrubias, M. M. Las proteinas
alimentacion. In Perspectiva de la Biotecnologia en Mexico; Fundacion
Javier Barrios Sierra. A.C. CONACYT. R.: Quintero, Mexico, 1985;
pp331350.
(16) Segura-Nieto, M.; Barba de la Rosa, A. P.; Paredes-Lopez, O.
Biochemistry of amaranth proteins. In Amaranth Biology, Chemistry
and Technology; Paredes-Lopez, O., Ed.; CRC Press: Boca Raton, FL,
1999; pp7695.
(17) Shewry, P. R. Cereal seed storage proteins: structures, properties
and role in grain utilization. J. Exp. Bot. 2002, 53, 947958.
(18) Derbyshire, E.; Wright, D. J.; Roulter, D. Legumin and vicilin,
storage proteins of legume seeds. Phytochemistry 1976, 15, 324.
(19) Shewry, P. R. Plant storage proteins. Biol. Rev. 1995, 70, 375
426.
(20) Nadal, P.; Canela, P.; Katakis, I.; OSullivan, K. Extraction,
isolation, and characterization of globulin proteins from Lupinus albus.
J. Agric. Food Chem. 2011, 59, 27522758.
(21) Association of Ocial Analytical Chemists (AOAC). Ocial
Methods of Analysis, 15th ed.; Washington, DC, 1990.
(22) Osborne, T. B. The Vegetable Proteins; Longmans: Green, NY,
1924.
(23) Barba de la Rosa, A. P.; Herrera, A.; Utsumi, S.; Paredes-Lopez,
O. Molecular characterization, cloning and structural analysis of a
cDNA encoding an amaranth globulin. J. Plant Physiol. 1996, 149,
527532.
(24) Chen, S.; Paredes-Lopez, O. Isolation and characterization of
the 11S globulin from amaranth seeds. J. Food Biochem 1997, 22, 53
65.
(25) Laemmli, U. K. Cleavage of structural proteins during the
assembly of the head of bacteriophage T4. Nature 1970, 217, 680
685.
(26) Shagger; Von Jagow, G. Tricin-sodium dodecyl sulfatepolyacrylamide gel electrophoresis for separation of protein in the
range from 1 to 100 kDa. Anal. Biochem. 1987, 166, 368379.
(27) Bello-Perez, L. A.; Agama-Acevedo, E.; Sanchez-Hernandez, L.;
Paredes-Lopez, O. Isolation and partial characterization of banana
starches. J. Agric. Food Chem. 1999, 47, 854857.
(28) Bienvenut, W. V.; Sanchez, J. C.; Karmime, A.; Rougue, V.;
Rose, K.; Binz, P. A.; Hochstrasser, D. F. Toward a clinical molecular
scanner for proteome research: parallel protein chemical processing
before and during western blot. Anal. Chem. 1999, 71, 48004807.

AUTHOR INFORMATION

Corresponding Author

*Phone: 01 (462) 6239674. E-mail:oparedes@ira.cinvestav.mx.

Funding

We thank the Consejo Nacional de Ciencia y Tecnolog aMexico for partial funding of this study and the scholarship to
M.R.S.-O.
Notes

The authors declare no competing nancial interest.

ACKNOWLEDGMENTS

ABBREVIATIONS USED

REFERENCES

We thank with pleasure Dr. Guillermo Mendoza-Hernandez

(FM, UNAM), Ing. Francisco Garc a Suarez (Ceprobi, IPN),
Dr. Luis E. Gonzalez de la Vara, and Dra. Mar a Elena Valverde
(Cinvestav, Irapuato) for very valuable technical advice and for
many critiques and helpful suggestions throughout this work.

BCA, bicinchoninic acid; CAD, collisionally activated dissociation; CS, chemical score; DSC, dierential scanning
calorimeter; DTT, dithiothreitol; EMS, enhanced mass
spectrometry; ESI, electrospray ionization; IDA, informationdependent acquisition; LC, liquid chromatography; ME, mercaptoethanol; MS, mass spectrometry; MW, molecular
weight; SD, standard deviation; TCA, trichloroacetic acid

(1) Cahill, J. P. Genetic diversity among varieties of chia (Salvia

hispanica L.). Genet. Res. Crop. Evol. 2004, 51, 773781.
(2) Ayerza, R.; Coates, W. Ground chia seed and chia oil effects on
plasma lipids and fatty acids in the rat. Nutr. Res. (N.Y.) 2005, 25,
9951003.
(3) Bueno, M.; Di Sapio, O.; Barolo, M.; Busilacchi, H.; Quiroga, M.;
Severin, C. Analisis de la calidad de los fruto de Salvia hispanica L.
(Lamiaceae) comercializados en la ciudad de Rosario (Santa Fe,
Argentina). Bol. Latinoam. Caribe Plantas Med. Aromaticas 2010, 9,
221227.
(4) Weber, C. W.; Gentry, H. S.; Kohlhepp, E. A.; McCrohan, P. R.
The nutritional and chemical evaluation of chia seeds. Ecol. Food Nutr.
1991, 26, 119125.
(5) Olivos-Lugo, B. L.; Valdivia-Lopez, M.A .; Tecante, A. Thermal
and physicochemical porpieties and nutritional value of the protein
fraction of Mexican chia seed (Salvia hispanica L.). Food Sci. Technol.
Int. 2010, 1, 18.
(6) Vazquez-Ovando, A.; Rosado-Rubio, G.; Chel-Guerrero, L.;
Betancur-Ancona, D. Dry processing of chia (Salvia hispanica L.) flour:
chemical characterization of fiber and protein. J. Food 2010, 8, 117
127.
(7) Ixtania, V.; Martnez, M.; Sportorno, V.; Mateo, M.; Maestri, D.;
Diehl, B.; Nolasco, S.; Tomas, M. Characterization of chia seed oils by
pressing and solvent extraction. J. Food Compos. Anal. 2011, 24, 166
174.
200

dx.doi.org/10.1021/jf3034978 | J. Agric. Food Chem. 2013, 61, 193201

Journal of Agricultural and Food Chemistry

Article

absorption and fluorescence techniques. Int. J. Pept. Protein Res. 1990,

35, 2534.

(29) Bidlingmeyer, B. A.; Cohen, S. A.; Tarvin, T. L. Rapid analysis of

amino acids using pre-column derivatization. J. Chromatogr. 1984, 336,
93104.
(30) Hendrikson, R. L.; Meridith, S. C. Amino acid analysis by
reverse-phase high performance liquid chromatography: precolumn
derivatization with phenylisothiocyanate. Anal. Biochem. 1984, 136,
6574.
(31) FAO/WHO/UNU. Protein and amino acids requirements in
human nutrition. WHO Tech. Report Ser. 2008, No. 935, 135183,
247248.
(32) Hsu, H.; Vavak, D.; Satterlee, L.; Miller, G. A. Multienzime
technique for estimating protein digestibility. J. Food Sci. 1977, 42,
12691279.
(33) Reyes-Caudillo, E.; Tecante, A.; Valdivia-Lopez, M. A. Dietary
fiber content and antioxidant activity of phenolic compounds present
in Mexican chia (Salvia hispanica L.) seeds. Food Chem. 2008, 107,
656663.
(34) Devinder, D.; Mona, M.; Hradesh, R.; Patil, R. Dietary fiber in
foods: a review. J. Food Sci. Technol. 2011, 49, 6369.
(35) Mosse, J. Nitrogen to protein conversion factor for ten cereals
and six legumes or oilseeds. A reappraisal of its definition and
determination. Variation according to species and to seed protein
content. J. Agric. Food Chem. 1990, 38, 1824.
(36) Aguirre, C.; Torres, I.; Mendoza-Hernandez, G.; Garca-Gasca,
T.; Blanco-Labra, A. Analysis of protein fractions and some minerals
present in chan (Hypstys suaveolens L.) seeds. J. Food Sci. 2011, 71,
1519.
(37) Nikokyris, P. N.; Kandylis, K. Feed protein fractions in various
solvents of ruminant feedstuffs. J. Agric. Food Chem. 1997, 75, 198
204.
(38) Fukushima, D. Structures of plant storage proteins and their
function. Food Rev. Int. 1991, 7, 353381.
(39) Plietz, P.; Damaschun, G.; Zirwer, D.; Gast, K.; Schwenke, K.
D.; Prakash, V. Shape and quaternary structure of -globulin from
sesame (Sesanum indicum L.) seeds as revealed by small angle X-ray
scattering and quasi-elastic light scattering. J. Biol. Chem. 1986, 261,
1268612691.
(40) Casey, R.; Domoney, C.; Smith, A. M. Biochemistry and
molecular biology of seed products. In Peas: Genetics, Molecular Biology
and Biotechnology; Casey, R., Davies, D. R., Eds.; CAB International:
Wallingford, UK, 1993; pp 121163.
(41) Romero-Zepeda, H.; Paredes-Lopez, O. Isolation and characterization of amarantin, the 11S amaranth seed globulin. J. Food Biochem.
1996, 19, 329339.
(42) Mori, T.; Utsumi, S. Purification and properties of storage
proteins of broad bean. J. Agric. Food Chem. 1979, 43, 577583.
(43) Brooks, J. R.; Morr, C. V. Current aspects of soy protein
fractionation and nomenclature. J. Am. Oil Chem. Soc 1985, 62, 1347
1353.
(44) Harwalkar, V. R.; Ma, C. Study of thermal properties of oat
globulin by differential scanning calorimetry. J. Food Sci. 1987, 52,
394398.
(45) Scilingo, A. A.; Anon, M. C. Calorimetric study of soybeans
protein isolates: effect of calcium and thermal treatments. J. Agric. Food
Chem. 1996, 44, 37513756.
(46) Cheung, S. C. M.; Szeto, Y. T.; Benzie, I. F. F. Antioxidant
protection of edible oils. Plant Foods Hum. Nutr. 2007, 62, 3942.
(47) Paredes-Lopez, O. Saower proteins for food use. In
Development in Food Proteins, 7 ed.; Hudson, B. J. F., Ed.; Elsevier
Science: London, UK, 1991; pp 133.
(48) Betancur-Ancona, D.; Gallegos-Tintore, S.; Chel-Gerrero, L.
Wet-fractionation of Phaseolus lunatus seeds: partial characterization of
starch and protein. J. Sci. Food Agric. 2004, 84, 11931201.
(49) Siddhuraju, P.; Becker, K. Effect of various domestic processing
methods on anti-nutrients and in vitro protein and starch digestibility
of two indigenous varieties of Indian tribal pulse, Mucuna pruriens val.
utilis. J. Agric. Food Chem. 2001, 49, 30583067.
(50) Deshpande, S. S.; Damodaran, S. Conformational characteristics
of legume 7S globulins as revealed by circular dichroic, derivative U.V.
201

dx.doi.org/10.1021/jf3034978 | J. Agric. Food Chem. 2013, 61, 193201